Project Summary Coronary vascular disease is the leading cause of death in the US and worldwide. Bypass grafts, utilized to circumvent blocked vessels, are a principle surgical treatment; however, intimal hyperplasia and vessel failure remain significant problems due to an inflammatory cascade stemming from tissue injury. Instructive biomaterials that attenuate maladaptive inflammatory responses and encourage adaptation to altered hemodynamic conditions would have significant clinical impact resulting in decreased procedure failure rates and a potential increase in the ability to use pre-treated autologous vessels for transplantation. Thus, our long- term goal is to develop biomaterials with desirable mechanical properties that can be easily placed on the exterior of at-risk venous grafts to beneficially direct the inflammatory process and provide a biological substrate for the formation of healthy neo-adventitium. The central hypothesis guiding our work is that engineered materials will guide inflammatory responses from pro-inflammatory to pro-healing phenotypes and encapsulated stem cells will further potentiate beneficial phenotypes. In Aim 1, we will thoroughly examine the effects of mechanical properties of PEG hydrogels on THP-1 monocyte-derived macrophages and CD14+ cells, isolated from both umbilical cord blood and peripheral blood, recruitment and polarization. We will evaluate cell proliferation and molecular phenotype in our constructs to gain insight into modulus/phenotype correlations and interrogate mechanisms for the observed effects. We anticipate that softer hydrogels will influence THP-1 macrophages and CD14+ cells towards a pro- inflammatory phenotype on softer hydrogels, while directing inflammatory cells towards a pro-healing and angiogenic phenotype on stiffer substrates, as a result of integrin mediated signaling. Next, in Aim 2, we will thoroughly interrogate the effects of human CD133+ cells and rabbit peripheral blood stem cells encapsulated within our established hydrogel formulations, and evaluate the impact of co-culturing human CD14+ and CD34+ cells on molecular phenotype, proliferation, and inflammatory, matrix remodeling, and regenerative biomolecule production to determine co-culture correlations and interrogate mechanisms for the observed effects. We expect that culture of CD14+ monocytes with CD133+-encapsulated gels will influence CD14+ cells to a less-inflammatory phenotype as a result of CD133+ paracrine secretions and direct CD14+ monocytes towards a pro-healing phenotype. Finally, animal models will be employed to evaluate target vessel responses to implanted hydrogels in vivo, in AIM 3. Hydrogels will be placed on the surface of skeletonized veins, and a series of experiments will be carried out to assess the effects of hydrogel composition and encapsulated cells on vessel anatomy. MicroCT and histological assessments will be performed at the termination of experiments to analyze the fine structure of vessels. We anticipate that optimized hydrogels will drive result in the expansion of the adventitium.